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United States Department of Agriculture

Agricultural Research Service

Related Topics

Research Project: BIOLOGY, EPIDEMIOLOGY, PATHOGENESIS, AND VECTOR SPECIFICITY OF SUGARBEET AND VEGETABLE VIRUSES

Location: Crop Improvement and Protection Research

2008 Annual Report


1a.Objectives (from AD-416)
Study vector transmission specificity, biology, epidemiology, detection and management of whitefly-transmitted criniviruses. Address pathogenicity and infection physiology of BNYVV and other soil-borne viruses of sugarbeet. Develop virus-induced gene silencing for control of curtoviruses in tomato and sugarbeet. Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management.


1b.Approach (from AD-416)
Evaluate factors contributing to the specificity of crinivirus transmission by whitefly vectors. Insect proteins will be separated and tested for interaction with whole virus and individual and combinations of virus proteins expressed in vitro. We will also conduct genetic and biological characterization of the criniviruses Lettuce chlorosis virus and Cucurbit yellow stunting disorder virus, and develop improved methods for detection and differentiation of criniviruses. Examine virus-host interactions, including differential protein expression and pathway activation in healthy sugarbeet and in sugarbeet infected with BNYVV, the causal agent of rhizomania. Studies will involve fractionation and separation of proteins and protein-protein binding studies. Attempt to develop infectious clones of BNYVV and BSBMV, and use these for generation of recombinant and pseudo-recombinant viruses that elucidate the viral genetic components responsible for BNYVV pathogenicity in sugarbeet, and increased disease severity during co-infection. Monitor for the emergence of BNYVV variants capable of overcoming known sources of resistance throughout the US beet industry using standard methods developed previously by our laboratory, and develop new methods for differentiation of resistance breaking isolates. Gene silencing constructs will be designed for control of curtoviruses in tomato and sugarbeet. Silencing constructs will be delivered in testing using either a virus-based vector carrying silencing constructs, or by delivery using Agrobacterium tumefaciens, and will be expressed as small interfering RNAs (siRNA). Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. This will involve biological, molecular and serological analyses including development of rapid detection tools, genetic characterization, vector identification and identification of factors contributing to virus emergence. (IBC info pending). Replaces 5305-22000-010-00D (3/07).


3.Progress Report
The Salinas lab maintains a collection of bipartite, whitefly-transmitted Criniviruses, and is recognized internationally as a leader in the study of this group of viruses. The lab has identified and characterized and most of the criniviruses that are currently known to infect sugarbeet and vegetable crops, and has developed detection methods for most known criniviruses. Studies recently completed (in press) have demonstrated that competition between viruses impacts the efficiency of crinivirus transmission differently depending on the host plant. In the fall of 2006 a new crinivirus, Cucurbit yellow stunting disorder virus (CYSDV) emerged for the first time in the US Desert Southwest. Our program has been actively assisting other laboratories in acquiring the tools to monitor for this virus, working with grower organizations, extension personnel, other scientists and regulatory authorities in determining the extent of infection, educating growers and developing management tactics to minimize losses.

Beet necrotic yellow vein virus (BNYVV) which causes rhizomania , and its vector, the soil-borne fungus, Polymyxa betae, were detected for the first time in the Western Hemisphere by the Salinas Virology Lab. Research on BNYVV and related viruses has been critical to understanding of the disease and facilitating development by the lab of highly specific and sensitive diagnostic assays. Results from studies of soil-borne sugarbeet viruses led to taxonomic reclassification of some viruses. The laboratory recently described a new strain of BNYVV that overcomes Rz1 gene resistance, that differs from other resistance-breaking (RB) strains by having only the standard 4 RNAs, rather than a fifth as is present in RB strains from Europe and Asia. Recent work by the Salinas Lab has linked the RB trait to amino acid changes in RNA3. The Salinas lab has also described two other soilborne viruses of sugarbeet (BSBMV and BOLV) that compete and interact with BNYVV under field conditions. Additional studies have genetically characterized soilborne viruses of lettuce associated with the diseases, lettuce dieback (two tombusviruses, one described by ARS Salinas) and lettuce big vein (Mirafiori lettuce big vein virus; MLBVV), and examined epidemiological factors contributing to control and spread. Collaborative efforts with sugarbeet and vegetable breeding programs in Salinas have facilitated development of genetic resistance to all three soilborne viruses (BNYVV, Tombusviruses and MLBVV).

Curly top, transmitted by the beet leafhopper (Circulifer tenellus) and caused by Beet curly top virus (BCTV) and related curtovirus members of the genus, Curtovirus, has impacted yields of sugar beet and vegetables since the late 1800s. The Salinas lab has identified crop and weed reservoirs, characterized changes in curtovirus population structure and determined this is likely influenced by environmental and cropping changes, and is developing novel methods for control.

This relates to NP 303, Component 4.


4.Accomplishments
1. Epidemiology of Cucurbit yellow stunting disorder virus (CSYDV) in the American Desert Southwest, and Identification of CYSDV in Florida. In the fall of 2006, CYSDV was first identified in California, Arizona and adjacent areas of Sonora, Mexico, and the USDA-ARS Virology Lab in Salinas, CA recently identified the virus in Florida melons in 2007. The lab is monitoring spring and fall crops for CYSDV incidence in crops and native weeds, and working closely with state and county personnel to examine incidence over several seasons. Research demonstrated CYSDV has a broader host range than was previously believed, and initial results indicated high infection in fall crops, and lower incidence in spring, with incidence directly correlated with increasing vector populations. The Virology Lab works closely with the melon breeding program at the USDA-ARS in Salinas, CA in evaluating a putative new source of resistance in melon, and is actively involved in educating growers and developing management tactics to minimize losses. All research described above is being conducted by the USDA-ARS Virology Lab in Salinas, in and supports NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2C: Population Dynamics, Spread, and Epidemiology of Pathogens.

2. BNYVV protein expression: a component of a proteomic analysis of resistance and susceptibility of sugarbeet to Beet necrotic yellow vein virus (BNYVV). New studies are using subtractive proteomics to identify proteins induced in resistant and susceptible sugarbeet (Beta vulgaris) as a result of infection with BNYVV. Studies conducted at the Sugarbeet Research Unit at the USDA-ARS in Ft. Collins, CO in collaboration with the Virology Lab at the USDA-ARS in Salinas, CA have identified differences in protein expression among healthy sugarbeet, sugarbeet exposed to the virus-free vector of BNYVV (Polymyxa betae), and BNYVV infected sugarbeet, as well as between resistant and susceptible sugarbeet. BNYVV proteins have been expressed and labeled in vitro, and protein interaction arrays are in progress to identify functional interactions between the virus and host plant. Results should elucidate physiological and biochemical changes that differ between healthy and BNYVV infected sugarbeet, leading to targeted methods to prevent BNYVV from eliciting symptoms of rhizomania disease. This project supports NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2B: Plant-Microbe-Vector Interactions.

3. Crinivirus transmission is influenced by host plant and altered by competition by a co-infecting virus. The criniviruses, Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus(TICV), share a common vector (greenhouse whitefly [T. vaporariorum]) and several host plants; however,ToCV can also be transmitted by other whitefly species. We have demonstrated that competition between these viruses impacts the efficiency of crinivirus transmission differently depending on the host plant, and this alters efficiency of transmission by the common vector in ways that may significantly impact epidemiology in the field. Follow-up work has resulted in in vitro expression of select viral proteins from both viruses for ongoing studies on vector specificity. Results will lead to new knowledge of what drives vector specificity of criniviruses, an emerging group of plant viruses worldwide, and determine factors contributing to the ability of these viruses to establish in an environment if introduced through international movement of plant material or other means. All work was conducted by the USDA-ARS Virology Lab in Salinas. This project supports NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2B: Plant-Microbe-Vector Interactions.

4. Development of efficient molecular detection of Lettuce chlorosis virus. Lettuce chlorosis virus (LCV), family Closteroviridae, genus Crinivirus, emerged as a threat to lettuce and sugar beet production in the desert regions of the southwestern U.S. in the early 1990’s, producing interveinal yellowing, stunting, and brittleness of affected leaves. The USDA-ARS Virology Lab in Salinas developed molecular probes and LCV-specific RT-PCR primer pairs, and demonstrated their efficacy for rapid identification of LCV infected lettuce and sugarbeet plants. Molecular probes have proven far more reliable than antiserum for crinivirus identification, and with the efficiency and reliability of our current molecular methods the value of serological detection for this virus is of lower importance and will not be continued. Results of our studies have provided industry with the diagnostic tools necessary for early identification, facilitating more effective disease control and decreasing losses. All work was performed by the USDA-ARS Virology Lab in Salinas, CA, and supports NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen.

5. Development of novel sources of resistance to Beet severe curly top virus (BSCTV) in sugarbeet and tomato. BSCTV and related curtoviruses are responsible for severe losses in numerous crops each year, including tomato and sugarbeet. Studies focused on development of virus induced gene silencing (VIGS) to obtain complete resistance against BSCTV and other curtoviruses in tomato and sugarbeet, and have identified genetic constructs capable of significantly reducing virus concentration and symptoms severity in replicated experiments. Downstream studies involving plant transformation with effective VIGS constructs are in progress to determine if more stable expression may optimize performance. Additional studies have been initiated toward obtaining complete resistance in both crops. The ability to elicit VIGS for control of curtoviruses will provide the vegetable and sugarbeet industries with alternative and potentially more effective control methods, reducing the need for excessive pesticide application. All work performed by the USDA-ARS Virology Lab in Salinas, CA. This project supports NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen.

6. Geographical and biological variation among curtovirus species affecting sugarbeet in the western United States. Losses due to curly top in sugarbeet have been increasing throughout the Rocky Mountain production region in recent years, suggesting the possible emergence of a new severe curtovirus species or strain. Sugarbeet plants exhibiting curly top symptoms were collected from throughout not only this region, but all affected sugarbeet production regions, and genus-specific and species-specific PCR primers were used for amplification and sequencing of viral genomic regions previously shown to be reliable for species differentiation. Sequence comparisons among isolates demonstrated that the dominant species have not changed substantially since a previous survey in the mid-1990s, but sequence conservation and divergence analysis indicated 3 different introductions to the U.S. based on differences in conservation within isolates of each of the 3 predominant species. This work illustrates that factors other than emergence of a new species is responsible for increasing disease problems, and clarifies curtovirus epidemiology. This project is a collaborative effort between the USDA-ARS Virology Lab in Salinas, and the NWISRL Pathology Lab in Kimberly, ID, and supports NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2C: Population Dynamics, Spread, and Epidemiology of Pathogens.

7. The Suppression of resistance-breaking Beet necrotic yellow vein virus isolates by Beet oak-leaf virus in sugar beet. Rhizomania, a serious disease of sugar beet (Beta vulgaris), is caused by Beet necrotic yellow vein virus (BNYVV), and the resistance allele Rz1 has been widely incorporated into commercial cultivars. Recently, resistance-breaking isolates of BNYVV (RB-BNYVV) were identified and characterized from throughout the sugar beet growing areas in the United States, and most soil samples and plants contained Beet oak-leaf virus (BOLV) as well as RB-BNYVV. Plants grown in soils infested with aviruliferous P. betae or carrying RB-BNYVVand BOLV, alone and in combination, were compared with plants grown in noninfested soil for differences in plant fresh weight and virus content to determine if interactions occur between these two viruses. Rz1 and Rz2 resistance genes that condition resistance to BNYVV did not confer resistance to BOLV, and results indicate that BOLV may suppress BNYVV in mixed infections. This work is important toward understanding factors influencing the ability of BNYVV to overcome sources of resistance as well as understanding factors contributing to rhizomania of sugarbeet. All research described above is being conducted by the USDA-ARS Virology Lab in Salinas, in support of NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statements 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen, and 2C: Population Dynamics, Spread, and Epidemiology of Pathogens.

8. Release of C812-41 and C812-41CMS Sugarbeet Germplasms with Resistance to Resistance-Breaking Strains of Beet Necrotic Yellow Vein Virus (RB-BNYVV). There is a need to develop sugarbeet germplasm with resistance to RB-BNYVV. Sugarbeet germplasm lines C812-41 and C812-41CMS were developed jointly by the Sugarbeet Breeding and Plant Virology Labs at the USDA-ARS in Salinas and released. C812-41 and its cytoplasmic male sterile counterpart have resistance to RB-BNYVV that causes the severe disease rhizomania. Resistance to RB-BNYVV was introgressed from wild beet Beta vulgaris subsp. maritima into monogerm C812-41, and although not yet confirmed, is suspected to be the Rz2 gene originating from WB42. This resistance was discovered in the germplasm enhancement program at Salinas and confers resistance to RB-BNYVV that defeats widely used Rz1. These germplasm resources represent ongoing efforts to combine multiple disease resistance with high productivity and to enhance source populations with genes from wild beet accessions, and this germplasm line will give the sugarbeet seed industry and ultimately the growers and processors an additional source of resistance to rhizomania. The virology research described above was conducted in support of NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen.

9. Specific polyclonal antibodies prepared against recombinant coat protein of Pelargonium zonate spot virus and immunodetection. Pelargonium zonate spot virus (PZSV) was identified in U.S. tomatoes in 2006, with infected plants showed stunting, malformation, yellow rings and line patterns on the leaves and concentric chlorotic ringspots on the stems. The coat protein (CP) gene of a California isolate of PZSV was amplified using primers designed to a published sequence, the amplified gene was cloned into an expression vector, with the resulting expressed protein tagged with 6xHis at the N-terminus. Recombinant CP, detected in the insoluble fraction, was purified and used to raise polyclonal antibodies. Crude antiserum was successfully used in indirect ELISA and Western blots to detect PZSV in infected tomato leaves as well as on a wide range of hosts, which will be of benefit in monitoring for this emerging tomato virus in tomato and other hosts. All research described above is being conducted by the USDA-ARS Virology Lab in Salinas, in support of NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen.

10. Analysis of distribution of Beet Black Scorch Virus (BBSV) in U.S., and develop methods for inoculation of BBSV. BBSV, initially characterized in China, is a new member of the genus Necrovirus and was first identified in a production field in Colorado in 2005. However, BBSV infected sugarbeet plants never showed the leaf symptoms described during the initial report; therefore, inoculation of the soil was a main concern. Rabbit anti-BBSV antiserum with high sensitivity was produced from a purified preparation of the virus. A soil survey for distribution of BBSV in sugarbeet production areas within the US was conducted by ARS scientists in the Crop Improvement and Protection Research Unit in Salinas, CA, and to date all of the soil samples from sugarbeet fields were negative for BBSV. Based on extremely low incidence in the U.S., and a questionable relationship between BBSV infection and previously described symptoms from China, continuation of research on inoculation methods is no longer a priority. Research described above was conducted by the USDA-ARS Virology Lab in Salinas, and supports NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen.

11. Genetic resistance to Cucurbit leaf crumple virus in melon. Cucurbit leaf crumple virus (CuLCrV), transmitted by Bemisia tabaci biotype B, is a begomovirus common in fall melons (Cucumis melo L.) planted from July through September in the American desert southwest. Melon breeding line MR-1, and six plant introductions (PI 124111, PI 124112, PI 179901, PI 234607, PI 313970, and PI 414723) exhibited partial resistance to CuLCrV in naturally infected field tests and controlled inoculation greenhouse tests. PI 236355 was completely resistant in two greenhouse tests, while other lines exhibited partial resistance as confirmed by PCR amplification of viral DNA. Genetic resistance to CuLCrV in melon involves a single, recessive gene. This resistance source will provide an important means of controlling this virus in an environment where other methods only slow the disease due to heavy vector pressure. This work is a collaboration between the USDA-ARS Virology and Vegetable Breeding Programs in Salinas, and the research described above is being conducted in support of NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen.

12. Biological and molecular characterization of an American sugar beet-infecting Beet western yellows virus isolate. Three aphid-transmitted viruses belonging to the Polerovirus genus, Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV), and Beet western yellows virus (BWYV), have been described as pathogens of sugar beet. Biological and serological characterization of an American sugar beet isolate of Beet western yellows virus (BWYV-USA) suggested that BWYV-USA displayed a host range similar to that of BMYV, but distinct from those of BChV and the lettuce and rape isolates of Turnip yellows virus. The complete genomic RNA sequence of BWYV-USA showed a genetic organization and expression typical of other Polerovirus members and provided indications of genetic relationships and possible evolutionary links to other members of the Polerovirus genus. Results demonstrate that BWYV-USA is a distinct species in the Polerovirus genus, and greatly clarify the nomenclature of this important group of viruses. This research was a collaborative effort between the USDA-ARS Virology Lab in Salinas, INRA, Colmar, France, and BBSRC, Broomsbarn, UK, and supports NP303 Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statements 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen, and 2C: Population Dynamics, Spread, and Epidemiology of Pathogens.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of Active CRADAs1
Number of Non-Peer Reviewed Presentations and Proceedings3
Number of Newspaper Articles and Other Presentations for Non-Science Audiences4

Review Publications
Liu, H., Lewellen, R.T. 2008. Suppression of Resistance-breaking Beet Necrotic Yellow Vein Virus Isolates by Beet Oak-leaf Virus in Sugar Beet.. Plant Disease 92:1043-1047

Larson, R.L., Wintermantel, W.M., Hill, A.L., Fortis, L.L., Nunez, A. 2008. Proteome changes in sugar beet in response to Beet necrotic yellow vein virus. Physiological and Molecular Plant Pathology. doi:10.1016/j.pmpp.2008.04.003

Polston, J.E., Hladky, L.L., Akad, F., Wintermantel, W.M. 2008. First Report of Cucurbit Yellow Stunting Disorder Virus in Cucurbits in Florida. Plant Disease. 92:1251

Wintermantel, W.M., Hladky, L.L. 2008. Resistance to curly top viruses through virus induced gene silencing. Phytopathology. 92:S172

Last Modified: 4/19/2014
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